Damos las gracias a K. Gill para obtener ayuda con la cría del ratón y el cuidado y la gestión de laboratorio. Gracias también a los miembros Mukoyama laboratorio para ayuda técnica. El financiamiento fue proporcionado por el Programa de Investigación Intramural del Instituto de Salud Naitonal.

1. Ratón recogida de la piel del miembro embrionarias (E13.5 E17.5 ~) <ol><li> Eutanasia conectado mujeres mediante el procedimiento de aprobación. De acuerdo con el protocolo aprobado animales, las hembras se sacrifican por el CO 2 de exposición y luego aseguró por dislocación cervical. Coloque el animal en su toalla de papel absorbente y disfrutar a fondo en el 70% de EtOH / H 2 O de un frasco. </li><li> Diseccionar el útero intacto y colocarlo en un 100 x 15 mm placa de Petri que contiene solución salina equilibrada de helado de Hanks (HBSS) para limpiarla de sangre. </li><li> Separados y diseccionar el embrión. Quitar el amnios muy delgada a partir del embrión. </li><li> (Opción) la disección de un solo embrión en un plato de 35 x 10 mm Petri si cada embrión debe ser genotipo. Cola disecados se transfiere a un tubo de PCR de 0,2 ml para el genotipado. </li><li> Cortar las extremidades anteriores del embrión y la transferencia de las extremidades anteriores con una pinza de anillo en 24 pocillos con 2 ml de helado de dulce 4% paraformaldehído (PFA) en tampón fosfato salino (PBS). </li><li> Fijar las patas delanteras con una suave mezcla en el mezclador Nutator a 4 ° C durante la noche. </li><li> Al día siguiente, retire la PFA y lavar tres veces durante 5 minutos en 2 ml de PBS con agitación suave en el mezclador de Nutator a temperatura ambiente. </li><li> Transferencia de las patas delanteras en el 100% de metanol (MeOH) y almacenarlas a -20 ° C congelador enzima (el congelador con el control de la temperatura crítica y sin función automática de descongelación). Anticuerpos primarios en la tabla 1 el trabajo después del tratamiento con MeOH al 100%. </li><li> La piel de fuera de la extremidad anterior con unas pinzas finas. La piel del miembro, cuando se deshidrata, deben separarse fácilmente de la extremidad. En primer lugar la extremidad con la cara ventral (lado de la palma) hacia arriba. Luego, usando unas pinzas finas, cortar la piel, como se muestra en el video. A continuación diseccionar alrededor de toda la extremidad, el pelado de la piel de suavidad sin ningún tipo de daño. </li></ol> 2. Todo el montaje la tinción inmunohistoquímica de Skins extremidades <ol><li> Rehidratar la piel de las extremidades en polipropileno de 5 ml de fondo redondo de tubo mediante la incubación a través de la serie graduada de MeOH / PBT (PBS + 0,2% Triton X-100) (75%, 50%, 25%) durante 5 minutos cada uno. Tenga en cuenta que el intercambio de soluciones debe tener cuidado para no dañar las pieles de las extremidades. </li><li> Lavar dos veces durante 5 min en PBT con una suave mezcla en el mezclador de Nutator a temperatura ambiente. </li><li> Bloquear la piel del miembro, ya sea con 10% de suero de cabra / PBS 0,2% TX100 tampón de bloqueo para detectar anticuerpos de cabra secundaria o el 10% de suero burro / PBS 0,2% TX100 tampón de bloqueo para detectar anticuerpos burro secundaria durante 2 horas con agitación suave en el mezclador en Nutator a temperatura ambiente. </li><li> Colocar las cáscaras en una extremidad 35 x 10 mm placa de Petri y la transferencia con una pinza de anillo en el tubo de 2 ml-microcentrífuga con 800μl de anticuerpos primarios (dilución adecuada como se indica en las tablas) en el tampón de bloqueo (ya sea de cabra 10% de suero / PBS 0,2 TX100% o 10% de suero de burro / PBS 0,2% TX100). Incubar las pieles de las extremidades con una suave mezcla en el mezclador de Nutator a 4 ° C durante la noche. Tenga en cuenta que varios anticuerpos primarios derivados de las diferentes especies (por ejemplo, el anticuerpo monoclonal de rata + anticuerpo policlonal de conejo) se pueden utilizar simultáneamente. </li><li> Al día siguiente, el lugar de la piel del miembro en un 35 x 10 mm placa de Petri y la transferencia con una pinza de anillo en polipropileno de 5 ml de fondo redondo de tubo con 4 ml de la solución de lavado (ya sea de cabra 2% de suero / PBS 0,2% o 2 TX100 % burro suero / PBS 0,2% TX100). </li><li> Lavar cinco veces durante 15 minutos con la mezcla suave en el mezclador de Nutator a temperatura ambiente. </li><li> Colocar las cáscaras en una extremidad 35 x 10 mm placa de Petri y la transferencia con una pinza de anillo en el tubo de 2 ml-microcentrífuga con 800μl de secundaria de anticuerpos en el amortiguador de bloqueo (ya sea de cabra 10% de suero / PBS 0,2% TX100 o el 10% burro suero / PBS 0,2% TX100). Normalmente se utiliza anticuerpos secundarios de Jackson ImmunoResearch (Cy3, Cy5, 1:300 diluciones) o Invitrogen (Alexa Fluor 488, Alexa Fluor 568, Alexa Fluor 633, 1:250 dilución). Filtrar la solución secundaria de anticuerpos con 0.22μm PVDF filtros de jeringa de membrana para eliminar las partículas de agregados de la secundaria de anticuerpos. Incubar la piel de las extremidades en la oscuridad o envuelto con papel de aluminio durante 1 hora con agitación suave en el mezclador de Nutator a temperatura ambiente. Tenga en cuenta que diferentes anticuerpos conjugados fluorescentes secundarios derivados de las diferentes especies se pueden utilizar simultáneamente. </li><li> Colocar las cáscaras en una extremidad 35 x 10 mm placa de Petri y la transferencia con una pinza de anillo en polipropileno de 5 ml de fondo redondo de tubo con 4 ml de la solución de lavado (ya sea de cabra 2% de suero / PBS 0,2% o 2% TX100 burro suero / PBS 0,2% TX100). Lavar cinco veces durante 15 minutos en la oscuridad o envuelto con papel de aluminio con mezclado suave en el mezclador de Nutator a temperatura ambiente. </li><li> (Opción para contratinción contra el núcleo) Incubar las pieles de las extremidades con 4 ml de la solución de lavado (ya sea de cabra 2% de suero / PBS 0,2% o 2% TX100 burro suero / PBS 0,2% TX100) con A-Pro-3 (Invitrogen T3605 , dilución 1:3000) en la oscuridad o envuelto con papel de aluminio durante 10 minutos con la mezcla suave en el mezclador de Nutator en la sala de temre. A continuación, lavar tres veces por 5 min con 4 ml de la solución de lavado (ya sea de cabra 2% de suero / PBS 0,2% o 2% TX100 burro suero / PBS 0,2% TX100) en la oscuridad o envuelto con papel de aluminio con mezcla suave el mezclador Nutator a temperatura ambiente. Tenga en cuenta que la tinción fuerte y específica para los núcleos que se observa para To-Pro-3 en una emisión específica (HeNe 633 nm de excitación). </li></ol> 3. Montaje de los Skins en la extremidad de diapositivas <ol><li> Colocar las cáscaras en una extremidad 35 x 10 mm plato de Petri. Eliminar el polvo, los cristales, las fibras de la capa interna de la piel con unas pinzas finas bajo el microscopio estereoscópico con poca iluminación para evitar la foto de un extenso descoloramiento. </li><li> Transferencia de las pieles de las extremidades para portaobjetos adhesivos con una pinza de anillo. Coloque las pieles con la capa interna mentir al alza en la diapositiva (es decir, hacia cubreobjetos). Aplanar la piel cuidadosamente con unas pinzas finas y eliminar traspaso de tampón de lavado por Kimwipe. </li><li> Monte en la lucha contra la decoloración medios de montaje sin burbujas de aire. Utilizamos 25 &quot;x25&quot; cubreobjetos. Cura en una superficie plana en la oscuridad (por ejemplo, las muestras montadas con prolongar el reactivo de oro se colocan durante la noche en la oscuridad a temperatura ambiente antes de ver). Para almacenamiento a largo plazo, sellar el cubreobjetos a la diapositiva y se almacenan a 4 ° C. </li></ol> 4. Microscopía Confocal <ol><li> Establecer láser adecuado para fluoróforos. Utilizamos microscopio Leica TCS SP5 confocal con tres fuentes láser de argón incluyendo 488nm (por Alexa Fluor 488 y GFP), 561nm DPSS (por Alexa Fluor 568 y Cy3) y HeNe 633 nm (por Alexa Fluor 633, Cy5 y To-Pro-3) . </li><li> Use la herramienta de búsqueda secuencial para evitar o reducir la interferencia en la que todos los colorantes en las muestras de doble o triple del manchar-se entusiasma al mismo tiempo. En el modo de exploración secuencial, las imágenes se grabarán en un orden secuencial. </li><li> Más información general sobre los tintes fluorescentes y láser para la excitación puede ser fundada en &quot;Microscopía Confocal de Biología&quot; por Hibbs (2004) </li></ol> 5. Resultados representante Todo el montaje immnofluorescence microscopía confocal de triple sello en la piel del ratón en el miembro anterior E15.5 con anticuerpos para el marcador de células endoteliales pan-PECAM-1 (Figura 1-C, D azul), el marcador de neurofilamentos 2H3 (Figura 1 verde), y las células de músculo liso marcador αSMA (Figura 1 A, B rojo) revela un patrón característico de ramificación de las arterias αSMA +, alineado con 2H3 + nervios periféricos en la piel del miembro. Además de la ramificación de los vasos sanguíneos, la piel esta extremidad modelo vascular se utiliza para estudiar los patrones de ramificación de vasos linfáticos con anticuerpos para el marcador de las células endoteliales linfáticas LYVE-1 (Figura 1 D, E de color rojo) y Neuropilin2 (NRP2) (Figura 1 D, F verde ). <img src="/files/ftp_upload/2620/2620fig1.jpg" alt="Figura 1" /> Figura 1. (AC) Las arterias se alinean con los nervios periféricos en la piel del miembro embrionario. Todo el montaje de triple sello microscopía confocal immnofluorescence con anticuerpos para el marcador de células endoteliales pan-PECAM-1 (AC, azul), el marcador de neurofilamentos 2H3 (A, verde), y el marcador de las células musculares lisas αSMA (A y B, de color rojo ) se muestra. En E15.5, 2H3 + nervios periféricos (flechas abiertas) asociados con las arterias (puntas de flecha), que están cubiertos por αSMA + en las células del músculo liso. (DF) linfático vascular en la piel del miembro embrionario. Triple-etiqueta de la microscopía confocal immnofluorescence con anticuerpos para el marcador de células endoteliales pan-PECAM-1 (D, azul), el marcador de las células endoteliales linfáticas LYVE-1 (D y E, de color rojo) y Neuropilin2 (NRP2) (D y F, verde ) se muestra. Los vasos linfáticos son visualizados por tanto LYVE-1 y NRP2, mientras que LYVE-1 también se expresa por una parte de los macrófagos (puntas de flecha abierta). Barra de escala: 100μm.

<ol>
	<li>Mukouyama, Y. S., Shin, D., Britsch, S., Taniguchi, M., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+nerves+determine+the+pattern+of+arterial+differentiation+and+blood+vessel+branching+in+the+skin.">Sensory nerves determine the pattern of arterial differentiation and blood vessel branching in the skin.</a> Cell. 109, 693-705 (2002).</li><li>Mukouyama, Y. S., Gerber, H. P., Ferrara, N., Gu, C., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+nerve-derived+VEGF+promotes+arterial+differentiation+via+neuropilin+1-mediated+positive+feedback.">Peripheral nerve-derived VEGF promotes arterial differentiation via neuropilin 1-mediated positive feedback.</a> Development. 132, 941-952 (2005).</li><li>Wang, H. U., Chen, Z. F., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+distinction+and+angiogenic+interaction+between+embryonic+arteries+and+veins+revealed+by+ephrin-B2+and+its+receptor+Eph-B4.">Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4.</a> Cell. 93, 741-753 (1998).</li><li>Gerety, S. S., Wang, H. U., Chen, Z. F., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symmetrical+mutant+phenotypes+of+the+receptor+EphB4+and+its+specific+transmembrane+ligand+ephrin-B2+in+cardiovascular+development.">Symmetrical mutant phenotypes of the receptor EphB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular development.</a> Mol Cell. 4, 403-414 (1999).</li></ol>

El sistema vascular es fundamental para el desarrollo de órganos durante la embriogénesis, así como para el mantenimiento de órganos y funciones de reproducción en los adultos, ya que proporciona suficiente oxígeno y nutrientes a los órganos. Red vascular adecuado es bien establecido, con procesos complejos y múltiples pasos por la angiogénesis en el que se reorganiza pre-existentes de la red capilar con estructuras muy ramificadas y jerárquica. A pesar de numerosos trabajos han demostrado que una variedad de ...

Anticuerpos Pan-marcador de las células endoteliales <table border="1"><tbody><tr><td> Anticuerpo </td><td> Especies </td><td> Empresa </td><td> Catálogo # </td><td> Condiciones de trabajo </td></tr><tr><td> PECAM-1 </td><td> Hamster Armenia (M) </td><td> Chemicon </td><td> MAB1398Z </td><td> Dilución 1:100 # 1 </td></tr><tr><td> PECAM-1 </td><td> Rata (M) </td><td> BD </td><td> 553369 </td><td> 1:300 dilución </td></tr><tr><td> VEGFR2 </td><td> Rata (M) </td><td> eBioscience </td><td> 14-5821-82 </td><td> Dilución 1:200 </td></tr><tr><td> CD34 </td><td> Rata (M) </td><td> eBioscience </td><td> 13-0341 </td><td> 1:300 dilución </td></tr><tr><td> Colágeno IV </td><td> Conejo (P) </td><td> AbD Serotec </td><td> 2150-1470 </td><td> 1:300 dilución # 2 </td></tr></tbody></table> Marcador de células endoteliales arteriales <table border="1"><tbody><tr><td> Anticuerpo </td><td> Especies </td><td> Empresa </td><td> Catálogo # </td><td> Condiciones de trabajo </td></tr><tr><td> Neuropilin1 </td><td> Conejo (P) </td><td> El laboratorio de la Kolodkin Alex # 3 </td><td></td><td> Dilución 1:3000 </td></tr><tr><td> Unc5H2 </td><td> Cabra (P) </td><td> I + D </td><td> AF1006 </td><td> Dilución 1:200 </td></tr></tbody></table> Venosa marcador de células endoteliales <table border="1"><tbody><tr><td> Anticuerpo </td><td> Especies </td><td> Empresa </td><td> Catálogo # </td><td> Condiciones de trabajo </td></tr><tr><td> EphB4 </td><td> Cabra (P) </td><td> I + D </td><td> AF446 </td><td> Dilución 1:100 </td></tr></tbody></table> Linfático marcador de células endoteliales <table border="1"><tbody><tr><td> Anticuerpo </td><td> Especies </td><td> Empresa </td><td> Catálogo # </td><td> Condiciones de trabajo </td></tr><tr><td> LYVE-1 # 4 </td><td> Conejo (P) </td><td> Abcam </td><td> ab14917 </td><td> Dilución 1:200 </td></tr><tr><td> LYVE-1 # 4 </td><td> Rata (M) </td><td> MBL </td><td> D225-3 </td><td> Dilución 1:200 </td></tr><tr><td> Prox-1 </td><td> Conejo (P) </td><td> Chemicon </td><td> AB5475 </td><td> Dilución 1:1000 </td></tr><tr><td> Prox-1 </td><td> Cabra (P) </td><td> I + D </td><td> AF2727 </td><td> Dilución 1:50 </td></tr><tr><td> Neuropilin2 </td><td> Conejo (P) </td><td> Señalización Celular </td><td> 3366 </td><td> 1, 100 de dilución </td></tr><tr><td> Podoplanin </td><td> Hámster sirio (M) </td><td> Banco hibridoma </td><td> 8.1.1 </td><td> Dilución 1:200 </td></tr></tbody></table> Células del músculo liso / pericitos marcador <table border="1"><tbody><tr><td> Anticuerpo </td><td> Especies </td><td> Empresa </td><td> Catálogo # </td><td> Condiciones de trabajo </td></tr><tr><td> αSMA-Cy3 </td><td> Ratón (M) # 5 </td><td> Sigma </td><td> c-6198 </td><td> Dilución 1:500 # 6 </td></tr><tr><td> NG2 </td><td> Conejo (P) </td><td> Chemicon </td><td> AB5320 </td><td> Dilución 1:200 </td></tr><tr><td> SM22α </td><td> Conejo (P) </td><td> Abcam </td><td> ab14106 </td><td> Dilución 1:200 </td></tr></tbody></table> Los anticuerpos para el reportero GFP <table border="1"><tbody><tr><td> Anticuerpo </td><td> Especies </td><td> Empresa </td><td> Catálogo # </td><td> Condiciones de trabajo </td></tr><tr><td> Las buenas prácticas agrarias </td><td> Conejo (P) </td><td> Invitrogen </td><td> A11122 </td><td> 1:300 dilución </td></tr><tr><td> Las buenas prácticas agrarias </td><td> Rata (M) </td><td> Nacalai Tesque </td><td> 04404-84 </td><td> Dilución 1:1000 </td></tr><tr><td> Las buenas prácticas agrarias </td><td> Pollo (P) </td><td> Chemicon </td><td> P42212 </td><td> 1:300 dilución </td></tr></tbody></table> Los anticuerpos para el reportero LacZ <table border="1"><tbody><tr><td> Anticuerpo </td><td> Especies </td><td> Empresa </td><td> Catálogo # </td><td> Condiciones de trabajo </td></tr><tr><td> β-gal </td><td> Conejo (P) </td><td> MP Biomédica </td><td> 55976 </td><td> Dilución 1:5000 </td></tr><tr><td> β-gal </td><td> Cabra (P) </td><td> AbD Serotec </td><td> 4600-1409 </td><td> Dilución 1:500 </td></tr><tr><td> β-gal </td><td> Pollo (P) </td><td> Abcam </td><td> ab9361 </td><td> Dilución 1:200 </td></tr></tbody></table> Los anticuerpos de los axones periféricos <table border="1"><tbody><tr><td> Anticuerpo </td><td> Especies </td><td> Empresa </td><td> Catálogo # </td><td> Condiciones de trabajo </td></tr><tr><td> 2H3 </td><td> Ratón (M) # 7 </td><td> Banco hibridoma </td><td> 2H3 </td><td> Dilución 1:200 </td></tr><tr><td> Tuj1 </td><td> Ratón (M) # 8 </td><td> Covance </td><td> MMS-435P </td><td> Dilución 1:500 </td></tr><tr><td> Periferina </td><td> Conejo (P) </td><td> Chemicon </td><td> AB1530 </td><td> Dilución 1:1000 </td></tr></tbody></table> Anticuerpos para la migración de las células de Schwann <table border="1"><tbody><tr><td> BFABP </td><td> Conejo (P) </td><td> El laboratorio de Thomas Müller es # 9 </td><td></td><td> Dilución 1:3000 </td></tr></tbody></table> (P): anticuerpo policlonal (M): anticuerpo monoclonal # 1: de cabra anti-armenio hámster-Cy3 (Jackson ImmunoResearch 127-165-160) de anticuerpos debe ser utilizado como anticuerpo secundario. # 2: Los anticuerpos colágeno IV puede ser utilizado para detectar los vasos sanguíneos después de la hibridación in situ. # 3: El anticuerpo es Neuropilin1 amablemente proporcionados por el laboratorio de la Kolodkin de Alex en la Universidad Johns Hopkins. De oveja anti-anticuerpos humanos Neuropilin1 está disponible en I + D (AF3870), aunque no lo he probado todavía. # 4: Los anticuerpos LYVE-1 también se detecta un subconjunto de los macrófagos en la piel embrionaria. # 5: El anticuerpo anti-ratón de anticuerpos es αSMA monoclonal IgG2a. # 6: El anticuerpo conjugado con Cy3 αSMA se incuba durante 1 hora a temperatura ambiente, junto con el secanticuerpos secundaria de otros anticuerpos primarios. # 7: 2H3 anticuerpo IgG1 monoclonal de ratón de anticuerpos contra neurofilamentos. # 8: Tuj1 anticuerpo monoclonal IgG2a de ratón de anticuerpos contra las neuronas específicas de la clase III beta-tubulina. # 9: El BFABP (cerebro-específica de ácidos grasos de unión a proteínas) de anticuerpos es amablemente proporcionados por el laboratorio Thomas Müller en Max-Delbrück-Center de Medicina Molecular.

whole-mount immunohistochemical analysis for embryonic limb skin vasculature: a model system to study vascular branching morphogenesis in embryo

Todo el montaje análisis inmunohistoquímico para obtener imágenes de todo el sistema vascular es fundamental para la comprensión de los mecanismos celulares de la ramificación morfogénesis. Hemos desarrollado el modelo de la piel del miembro vasculatura para estudiar el desarrollo vascular en el que un pre-existentes primitivo plexo capilar se reorganiza en una red jerárquica ramificada vascular. Todo el montaje de microscopía confocal con el etiquetado múltiple permite a la imagen robusta de los vasos sanguíneos intactos, así como sus componentes celulares incluyendo las células endoteliales, pericitos y células de músculo liso, utilizando marcadores fluorescentes específicos. Los avances en este modelo las extremidades vasculatura la piel con los estudios genéticos han permitido comprender los mecanismos moleculares del desarrollo vascular y el patrón. La piel del miembro vasculatura modelo ha sido utilizado para estudiar cómo los nervios periféricos proporcionan un modelo espacial para la diferenciación y el patrón de las arterias. En este artículo se describe un protocolo de vídeo sencillo y robusto para teñir los vasos sanguíneos intactos con vascular anticuerpos específicos y anticuerpos fluorescentes secundaria, que es aplicable para vascularizado órganos embrionarios en los que son capaces de seguir el proceso de desarrollo vascular.

Watch this Scientific Journal Video about Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo at JoVE.com

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Se introduce un inmunohistoquímica todo el montaje y la microscopía confocal de barrido láser con múltiples etiquetas para analizar la formación de complejos de la red vascular de la piel del ratón extremidades embrionarias.

Todo el montaje El análisis inmunohistoquímico de la piel del miembro Vasculatura embrionarias: un sistema modelo para estudiar la morfogénesis vascular ramificación en embriones

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

whole-mount-immunohistochemical-analysis-for-embryonic-limb-skin

Research

JoVE Journal

Biology

17.5K vistas.  National Heart, Lung, and Blood Institute, National Institutes of Health. Se introduce un inmunohistoquímica todo el montaje y la microscopía confocal de barrido láser con múltiples etiquetas para analizar la formación de complejos de la red vascular de la piel del ratón extremidades embrionarias.

Video: Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Monte doble entero hibridación in situ de embriones de pollo temprano

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Investigación

Biología

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Método para la tinción de anticuerpos enteros en el monte del polluelo

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Pez cebra todo el montaje de alta resolución doble fluorescente In Situ La hibridación

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

Cultura de embriones de ratón de pulmón, un sistema para evaluar los mecanismos moleculares de la ramificación

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Do-It-Yourself de dispositivos para la recuperación de las muestras criopreservadas de caída accidental en los tanques de almacenamiento criogénicos

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Un sistema para la<em&gt; Ex vivo</em&gt; Cultivo de páncreas embrionario

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

ARN<em&gt; In situ</emHibridación&gt; Los embriones en todo el montaje y la histología de células Adaptado para elasmobranquios marinos

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Embriones de ratón con el desarrollo en todo un sistema de cultivo de embriones sin suero

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Análisis de la apoptosis en embriones de pez cebra en Todo el montaje de inmunofluorescencia para detectar la caspasa 3 activada

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...